Stock No: 005813
Protocol 29194: Standard PCR Assay - Grik5<tm1Dgen> STD PCR
Version 2.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = 425 bp
Heterozygote = 425 bp and 550 bp
Wild type = 550 bp
Add 2&micro;l Cresol Red to each sample post PCR.<br /> Separate on a 1.5% electrophoesis gel

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
moIMR0012 GGG TGG GAT TAG ATA AAT GCC TGC TCT A
oIMR5002 GGT TGT ACC ACT CAT AAG GGC TCA GC A
oIMR5003 GGT ACT ATG GCT TCT CAG TTC CTG G A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
moIMR0012 0.50 uM
oIMR5002 0.50 uM
oIMR5003 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.